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A. Falgàs et al.
rate.2 Relapse after R-CHOP therapy occurs in 40% of patients;3,4 this is currently managed with salvage chemotherapy. This is followed by high-dose chemother- apy and autologous bone marrow transplant in patients with chemosensitive disease, which, however, leads to long-term disease control in only half of the patients.5 Moreover, less than 20% of patients treated with an R- CHOP front-line regimen who relapse within one year benefit from salvage autologous hematopoietic cell trans- plant.2,6 Thus, novel therapeutic strategies that reduce relapse rates and enhance DLBCL patient survival are urgently needed.
Novel approaches based on selective-drug delivery to
cancer cells promise to increase patient benefit by offering
both higher cure rates and lower side effects in DLBCL
patients. In this regard, we evaluated a previously devel-
oped protein nanocarrier as a possible drug carrier to pur-
sue the selective elimination of DLBCL cells over-express-
ing CXCR4 (CXCR4+), which are responsible for DLBCL
relapse and disease progression.7-9 Thus, the
CXCR4-CXCL12 axis is involved in tumor pathogenesis,
cancer cell survival, stem cell phenotype, and resistance to
chemotherapy.10,11 In addition, CXCR4 is constitutively
over-expressed in NHL cell lines,12,13 and also in approxi-
mately 50% of malignant B-cell lymphocytes derived
from DLBCL patients.8 Interestingly, CXCR4+ DLBCL cell
lines show resistance to rituximab but are sensitive to the
combination of rituximab with a CXCR4 antagonist.14,15
Most importantly, we and others reported that CXCR4
overexpression associates with poor progression-free and
overall survival in DLBCL patients treated with R- CHOP.7,8,14
Our group has developed T22-GFP-H6, a self-assem- bling protein nanocarrier, which uses the peptidic T22 ligand to target the CXCR4 receptor.16 This carrier displays a high recirculation time in blood and selectively biodistributes to tumor tissues in solid tumor models, internalizing selectively in CXCR4+ cancer cells, while increasing its tumor uptake compared to the untargeted GFP-H6 counterpart.17 This nanocarrier is also able to incorporate toxins (e.g. diphtheria toxin catalytic domain) leading to selective elimination of CXCR4+ colorectal can- cer cells.18,19 Nevertheless, no previous protein-based nanocarrier has been described to specifically target cancer cells in hematologic neoplasias. Critical differences between solid cancers and hematologic neoplasias may raise doubts about its use to target CXCR4+ cancer cells in DLBCL models. Thus, the enhanced permeability/reten- tion (EPR) effect, due to abnormal fenestrated vessels and limited lymphatic drainage, allows nanocarrier accumula- tion in solid tumors. In contrast, DLBCL is a disseminated disease that displays freely circulating lymphoma cells in blood concomitantly with their confinement at specific tumor niches, such as lymph nodes (LN) and bone mar- row (BM), in which the EPR effect is unlikely to be pres- ent.20
Here, we studied whether active targeting of the T22- GFP-H6 nanocarrier leads to its selective uptake in CXCR4+ subcutaneous (SC) DLBCL tumors. We also assessed if this increased uptake associates with specific nanocarrier internalization in CXCR4+ lymphoma cells;
21,22
issues still be to settled in nanomedicine. Importantly,
we used a disseminated CXCR4+ DLBCL model (which replicates the organ involvement observed in DLBCL patients8) to study nanocarrier accumulation in lym-
phoma-affected organs (LN and BM) and its capacity to internalize in CXCR4+ lymphoma cells within these organs. Moreover, we evaluated whether T22-DITOX- H6, a nanoparticle incorporating a diphtheria toxin domain that maintains the same structure as the nanocar- rier, can selectively eliminate CXCR4+ DLBCL cells in SC tumors. The study goal was to determine whether we could use the nanocarrier to selectively deliver drugs to target CXCR4+ DLBCL cells.
Methods
In vivo experiments
Four-week old female NOD/SCID mice were obtained from
Charles River Laboratories. Mice were maintained in specific pathogen-free (SPF) conditions with sterile food and water ad libitum. Mouse experiments were approved by the Hospital de la Santa Creu i Sant Pau Animal Ethics Committee.
For SC models, 10 million DLBCL cells were injected in both flanks. Tumor growth was monitored twice a week with a caliper (tumor volume=width2 x length/2). When tumors reached a volume of 600-800 mm3, mice received a single intra- venous (IV) dose of 200 mg T22-GFP-H6, which contains a fluo- rescent domain, or buffer (20 mM Tris, 500mM NaCl, pH 8). T22-GFP-H6 design and production have been described in pre- vious studies.16 Fluorescence intensity (FLI) was measured ex vivo at different time points in tumors, plasma, and all organs. A plas- ma pool was obtained by centrifugation of total blood, obtained by intracardiac puncture (25G), at 600g for ten minutes (min) at 4oC. T22-GFP-H6 biodistribution in SC tumors over time was measured using the area under the curve (AUC). AUC analysis of tumors and normal organs was measured using the GraphPad Prism 6 program. We subcutaneously administered AMD3100 in mice to perform CXCR4 blocking experiments, giving a total of three AMD3100 doses at 10 mg/kg, 1 hour (h) before and 1h and 2h after IV T22-GFP-H6 injection. We used SC tumor models to evaluate the antitumor effect and associated toxicity of T22- DITOX-H6. Mice received a single 25 mg IV dose of T22- DITOX-H6 or buffer when tumors reached a volume of 400-600 mm3. Animals were euthanized 24h post administration. T22- DITOX-H6 nanoparticle characterization has been published previously.18
To generate the disseminated lymphoma model, NOD/SCID mice were intravenously injected with 20x106 luminescent Toledo cells (Toledo-Luci) in 200 mL physiological serum. Dissemination was monitored capturing bioluminescence inten- sity (BLI) twice a week after intraperitoneal injection of firefly D-luciferin. After 27-30 days, animals received a single IV dose of 400 mg T22-GFP-H6 nanocarrier or buffer. Five hours later, FLI was measured ex vivo in all organs.
Fluorescence intensity correlates to the amount of accumulat- ed protein in each tissue and is expressed as average radiant effi- ciency. FLI from experimental mice was calculated subtracting the FLI auto-fluorescence of control mice. The emitted FLI and BLI were measured using the IVIS Spectrum 200 Imaging System (Xenogen). Finally, tumors and all organs were collected, fixed and paraffined to perform histological, immunohistochemical or immunofluorescent evaluations, and were also directly cryopre- served in liquid nitrogen for protein extraction.
Details of methods for cell culture, transfection with Luciferase and CXCR4 plasmids, cell proliferation, flow cytom- etry, western blot, histopathology, 4′,6-diamidino-2-phenylin- dole (DAPI) staining, immunohistochemistry (IHC) and immunofluorescence (IF) analyses can be found in the Online Supplementary Appendix.
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